Membrane-mimetic lipid films with or without cholesterol were formed on alkylsilane self-assembled monolayers on glass by a process of lipid vesicle fusion. Using a combination of radiochemical titration with C-14-labeled dipalmitoylphosphatidylcholine (DPPC) and AFM imaging in an aqueous environment, we have characterized both molecular packing density and, in an homologous series of lipid monolayers, film thickness. These data confirm that a monolayer phospholipid membrane can be produced by vesicle fusion to an alkylated glass substrate.